Abstract
Antimicrobial resistance (AMR) describes a decrease in the efficacy of antimicrobial drugs (e.g., antibiotics) against microbes (e.g., bacteria). AMR poses a significant clinical threat and was directly attributed to 1.28 million deaths in 2019 alone. New treatment options, such as novel antibiotics or antibiotic adjuvants, are urgently required to treat these resistant bacterial infections. Antibiotic adjuvants enhance the bactericidal activity of antibiotics, which can potentiate antibiotic efficacy against drug-resistant bacteria. According to the World Health Organisation (WHO), carbapenem- and cephalosporin-resistant Acinetobacter baumannii, Enterobacterales, and Pseudomonas aeruginosa are of especially high concern as treatment options for these antibiotic-resistant bacteria are quickly dwindling. At the same time, morbidity and mortality associated with these infections continue to rise. One drug which is currently being investigated as a broad-spectrum antibiotic adjuvant is zinc ionophore PBT2, which has previously been shown to resensitise Neisseria gonorrhoeae and Group A Streptococcus to polymyxins and Methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE) and Streptococcus pneumoniae to β-lactam antibiotics. It is hypothesised that antibiotic resensitisation (i.e., the potentiation of antibiotic efficacy in resistant bacteria) occurs due to the disruption of metal ion homeostasis brought on by PBT2-mediated Zn accumulation, which impacts downstream pathways involved in antibiotic resistance. However, the exact mechanism through which PBT2 increases antibiotic sensitivity across all these diverse pathogens remains unknown. Little study has focused on the role of PBT2, and more widely metal ion homeostasis, in the antibiotic resensitisation of Gram-negative bacterial species to antibiotics other than polymyxin, despite Gram-negative bacteria making up 9 of the top 11 critical and high-priority antibiotic-resistant bacterial pathogens identified by the WHO. To address this knowledge gap, we used PBT2 as a tool compound to better understand the role of metal ion homeostasis in breaking resistance to a range of clinically utilised antibiotics: meropenem, ceftriaxone, gentamicin, colistin, aztreonam, and ceftazidime in multi-drug-resistant (MDR) clinical isolates of Gram-negative bacteria: A. baumannii, P. aeruginosa, K. pneumoniae and E. coli. We observed PBT2-mediated
resensitisation of P. aeruginosa to ceftriaxone in nutrient-depleted conditions and resensitisation of E. coli to gentamicin in high-nutrient conditions. The variation in efficacy of PBT2 in mediating antibiotic resensitisation observed in this study against the tested isolates points towards species-specific efficacy. Upon further investigation, we uncovered that supplementation of 15 µg/mL PBT2 in MDR E. coli reduced the inhibitory concentration of gentamicin from >512 µg/mL to 4 µg/mL. In addition, we observed that PBT2 + gentamicin treatment caused an influx of zinc and a depletion of potassium and magnesium. Notably, we also observed a downregulation of aph3, which encodes an aminoglycoside-modifying enzyme, an upregulation of zntA (Zn exporter), copA (copper exporter) and fur (iron starvation-induced protein), an upregulation of porins (chiP) and a downregulation of multi-drug efflux component acrA in PBT2 + gentamicin-treated E. coli compared to untreated. These data suggest that PBT2 has a widespread effect on MDR E. coli. However, the species-specific responses observed in this study indicate that while PBT2 is a valuable tool compound for elucidating the underlying mechanisms of antibiotic potentiation, it is not a perfect solution to overcome AMR. The disruption of metal ion homeostasis appears to be a viable avenue for future development of antibiotic adjuvants; however, a more detailed understanding of why PBT2 only works in specific bacterial species for certain antibiotics is required. This understanding may allow these PBT2-impacted pathways to be more specifically targeted to design more efficacious antibiotic adjuvants or antibiotics. This study contributes to the growing body of evidence supporting intracellular metal ion regulation as a promising avenue for addressing AMR's immense challenges and highlights the potential of targeting metal ion homeostasis to restore antibiotic sensitivity in MDR bacterial pathogens.